The present invention relates to transgenic plants, in particular, the present invention relates to transgenic plants expressing a calcium-binding protein that increases available calcium stores in the plant.
Calcium plays an essential role in plant growth and development and is involved in multiple signal transduction pathways. Whereas cytoplasmic calcium concentrations are tightly regulated, higher levels of calcium are found in subcellular organelles (Gilroy et al., (1993) J. Cell Science 106:453). Modulation of cytoplasmic calcium levels provides a rapid response to environmental stimuli and is achieved by a system of Ca2+-transport and storage pathways that include Ca2+ buffering proteins in the lumen of intracellular compartments. The endoplasmic reticulum (ER), cell wall, and the vacuole contain high levels of calcium that could be released to the cytoplasm. Unlike animal cells, the majority of Ca2+ in plant cells is found in the cell wall and vacuole, not in the ER (Bush, (1995) Ann. Review Plant Physiol Plant Molec. Biol. 46:95). Except for the vacuole, which may not readily release calcium (Hirschi et al., (1999) Plant Cell 11:2113), the availability of these stores for signaling has not been demonstrated. A voltage-gated, calcium-release channel has been identified in the endoplasmic reticulum (ER) of plants (Klusener et al., (1995) EMBO J. 14:2708). This channel is responsive to mechanotransduction, suggesting that the ER calcium store may be an important component of signal transduction pathways in plants as well as animals (Klusener et al., (1995) EMBO J. 14:2708). In plants, the major Ca2+ storage protein in the ER is calreticulin (CRT) (Hassan et al., (1995) Biochem. Biophys. Res. Commun. 211:54; Navazio et al., (1998) Plant Physiol. 109:983).
Ca2+ has long been recognized as an important second messenger responsible for mediating the activities of many environmental and endogenous signals. Cytosolic Ca2+ concentrations often show significant changes in plant cells under the influence of various stress signals such as touch, cold or heat shock, wounding, anoxia, salinity, and hypoosmotic shock (Knight et al., (1991) Nature 352:524; Knight et al., (1992) Proc. Nat. Acad. Sci. USA 89:4967; Knight et al., (1996) Plant Cell 8:489; Haley et al., (1995) Proc. Nat Acad. Sci. USA 92:4124; Campbell et al., (1996) Cell Calcium 19:211; Polisensky and Braam, (1996) Plant Physiol. 11:1271; Subbaian et al., (1994) Plant Physiol. 105:369; Lynch et al., (1989) Plant Physiol. 90:1271; Bush, (1996) Planta 199:89; Okazaki et al., (1996) Plant, Cell and Environment 19:569; Takahashi et al., (1997) Plant Physiol. 113:587). A stress-induced change in cytoplasmic calcium concentrations may be one of the primary transduction mechanisms whereby gene expression and biochemical events are altered to adapt plant cells to environmental stresses (Monroy et al., (1993) Plant Physiol. 102:1227; Subbaiah et al., (1994) Plant Physiol. 105: 369; Monroy and Dhindsa, (1995) Plant Cell 7:321; Braam et al., (1996) Physiol. Plant 98:909).
A variety of plant diseases and growth disorders causing substantial losses to horticultural crops have been attributed to calcium deficiency. Color breakdown in Anthurium spathes (Higaki et al., (1980) J. Am. Soc. Hortic. Sci. 105;438; Higaki et al., (1980) J. Am. Soc. Hortic. Sci. 105:441) can result in field losses of 50% and losses after shipments of up to 20%. Other conditions include but are not limited to tipburn in lettuce, cabbage and cauliflower (Goto and Takakura, (1992) Trans. Am. Soc. Ag. Engineers 35;641; Barta and Tibbitts, (1986) J. Am. Soc. Hortic. Sci. 111:413; Aloni, (1986) J. Hortic. Sci. 61:509; Maynard et al., (1981) Hotsci. 16:193), shoot-tip necrosis in potatoes, a physiological disorder found in normal microculture conditions, that makes the cultures useless for micropropagation or research (Sha et al., (1985) J. Am. Soc. Hortic. Sci. 110:631); and blossom end rot in tomato (DeKock et al., (1980) J. Sci. Food Agric. 33:509; Banuelos et al., (1985) Am. Soc. Hortic. Sci. 20:894; Ho and Adams, (1994) J. Hortic. Sci. 69:367). Addition of CaCO3 does not remedy the problem in areas where additional factors such as soil salinity and pH are sub-optimal (Bower and Turk, (1946) J. Am. Soc. Agron. 38:723; McLaughlin et al., (1993) Can. J. For. Res. Rev. Can. Rech. For. 23:380; McCray et al., (1991) Soil Use Manage 7:193; Francois et al., (1991) Hort. Science 26:549) or where the condition results from localized deficiencies caused by uneven Ca2+ distribution in tissues (Francois, et al, (1991) Hort. Science 26:549; Ho and Adams, (1994) J. Hortic. Sci. 69:367). Deficiencies may be exaggerated by high transpiration rates in a desert environment or a reduction in root pressure resulting from soil salinity (Francois, et al, (1991) Hort. Science 26:549; Ho and Adams, (1994) J. Hortic. Sci. 69:367). A temporary calcium deficiency of 8-10 days resulted in reduced stem growth and death of the apical meristem in tomato (Morand et al., (1996) J. Plant Nutr. 19:115).
Calreticulin is a multifunctional calcium-binding protein that is highly conserved in eukaryotic cells (Michalak et al. (1998) Biochem. Cell. Biol. 76:779; Michalak et al., (1999) Biochem. J. 344 Pt. 2:281; Dresselhaus et al., (1996) Plant Molec. Biol. 31:23; Krause and Michalak, (1997) Cell 88:439). The conservation of CRT and the fact that CRT knockouts are lethal in mice (Mesaeli et al., (1999) J. Cell. Biol. 144:857) suggest that CRT performs an essential function. In plants, CRT has been localized to the endoplasmic reticulum, Golgi, plasmodesmata, and plasma membrane (Borisjuk et al., (1998) Planta 206:504; Hassan et al. (1995) Biochem. Biophys. Res. Commun. 211:54; Baluska et al., (1999) Plant J. 19:481). The protein includes a signal sequence and ER retention motif for ER localization, and also has a nuclear localization sequence. Although these localization sequences appear to be conserved across species, there is contradictory evidence for nuclear localization.
CRT has been shown to function as a chaperone in the ER (Peterson and Helenius, (1999) J. Cell. Sci. 112:2775; Saito et al., (1999) EMBO J. 18:6718; Denecke et al., (1995) Plant Cell 7:391; Nauseef et al., (1995) J. Biol. Chem. 270:4741; Qtteken and Moss, (1996) J. Biol. Chem. 271:97; Crofts and Denecke, (1998) Trends Plant Sci. 3:396). Other proposed roles include regulation of gene expression (Perrone et al. (1999) J. Biol. Chem. 274:4640; signaling (Rauch et al., (2000) Exp. Cell Res. 256:105), and serving as a calcium buffer (Mesaeli et al., (1999) J. Cell. Biol. 144:857). In animal cells, calreticulin mRNA decreases during calcium depletion, along with resting and IP3-sensitive calcium pools (Mailhot et al., (2000) Endocrinology 141:891). Persson et al. (in press) demonstrates that altered expression of calreticulin (CRT) altered Ca2+ uptake and release in ER-enriched membrane fractions. The data indicate that the pool of Ca2+ in the ER can be affected by altering expression of CRT.
CRT has three functional domains: a globular N-domain, a proline rich, high affinity, low capacity Ca2+-binding domain (the P-domain) and a highly acidic, low affinity, high capacity Ca2+-binding domain (the C-domain) (Michalak et al., (1992) Biochem. J. 285:681). The P-domain shares considerable homology with the ER chaperone calnexin, which is also found in plants and functions as a chaperone. In addition, in Xenopus oocytes the P-domain has been implicated as the active region in Ca2+ signal transduction (Camacho and Lechleiter, (1995) Cell 82:765). The C-domain is a highly acidic region that has been shown to bind 20-50 moles of Ca2+/mole of protein and, thus, appears to be a major site of Ca2+ storage within the ER (Michalak et al., (1992) Biochem. J. 285:681). Calsequestrin, a calcium-binding protein related to the C-domain of CRT, is not found in plants (Navazio et al., (1995) J. Eukeryot. Microbiol. 45:307).
Hirschi et al., (1999) Plant Cell 11:2113 over-expressed a tonoplast Ca2+/H+ antiporter (CAX) in Arabidopsis and tobacco. Plants transgenic for CAX had an increased need for calcium and showed a hightened sensitivity to cold when grown on normal medium. It appears that the introduction of the CAX transgene resulted in storage of calcium in a form that was not available to the plant, thereby producing a calcium-deficient state in the plant.
WO 98/36084 suggests transforming a plant with bovine intestinal calcium binding protein to increase calcium accumulation in the plant. However, this protein only binds 2 moles of calcium per mole of protein, and over-expression of this protein may have adverse consequences on calcium signaling and homeostasis in the plant. Further, it is unclear whether calcium bound to this protein would be in a biologically active form for the plant.
Accordingly, there is a need in the art for methods of improving available calcium stores in plants without unduly perturbing calcium homeostasis.
The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a C-domain fragment, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization signal peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant.
The inventive transgenic plants may have advantageous phenotypic traits as compared with wild-type plants. Increased calcium storage in the plants may result in an enhanced resistance (i.e., tolerance) to calcium-limiting conditions and/or improved stress resistance under such conditions. The plants of the invention may further have enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer supplementation. Further, the plants of the invention may have an enhanced nutritional value (e.g., as a source of calcium) for use as human food or animal feed.